Books - Non-fiction

Biomolecule Sensing with Adaptive Plasmonic Nanostructures

Description
... It is known that the aggregation of even a few particles makes a large difference for ... in DI water 5 times), (c) after incubation with antigen cocktail containing 1nM human interleuikin 10. ... of R6G molecules can potentially be applied to
Published
of 16
All materials on our website are shared by users. If you have any questions about copyright issues, please report us to resolve them. We are always happy to assist you.
Related Documents
Share
Transcript
  Biomolecule Sensing with Adaptive PlasmonicNanostructures Vladimir P. Drachev and Vladimir M. Shalaev Purdue University, West Lafayette, IN 47907 { vdrachev,shalaev } @purdue.edu 1 Introduction One of the challenges of biomolecule sensing with surface-enhanced Ramanscattering (SERS) is to preserve all of the advantages of Raman spectroscopyapplications for structural biology. There are many examples where Ramanspectroscopy provides important information on large, macromolecular struc-tures as a whole and in defining small regions of large complexes throughligand–macromolecule recognition (reviewed in [1, 2, 3, 4, 5, 6]). The Ramanscattering process involves interplay between atomic positions, electron dis-tribution, and intermolecular forces. Hence, Raman spectroscopy potentiallycan be one of the techniques used to reveal correlations between structureand function.It is a common belief that the protein–metal surface interaction maylead to structural changes of proteins and the loss of protein functionalityto some extent. To what extent this occurs is a question that needs to beaddressed for any particular type of SERS-active substrates. The applicabil-ity of SERS to molecular biology has been under extensive study since the1980s [7]. Despite some unknowns in the SERS process, the molecular mecha-nisms of biomolecule–metal surface interactions and the distance dependenceof the Raman enhancement, SERS has been widely used in biomolecularspectroscopy [8,9,10,11,12,13,14,15,16].In this Chapter we demonstrate several examples of protein sensing withour SERS substrate employing a new, adaptive property of well-known vac-uum-evaporated silver films. The deposition of protein solutions on such afilm results in the rearrangement of the initial metal nanostructures. Suchprotein-mediated restructuring leads to the formation of aggregates of metalparticles naturally covered and matched with molecules of particular sizesand shapes. This procedure optimizes the SERS signal and, in parallel, stabi-lizes the metal surface with proteins. Such a substrate, which is referred to asan  adaptive silver film (ASF) , provides a large SERS enhancement and henceallows protein sensing at monolayer protein surface densities, while enablingthe adsorption of proteins without significant changes in their conformationalstates [17, 18, 19, 20]. For example, we showed that spectral differences inSERS spectra of human insulin and its analog insulin lispro can be detectedand assigned to their difference in conformational states. An interesting op- K. Kneipp, M. Moskovits, H. Kneipp (Eds.): Surface-Enhanced Raman Scattering – Physics andApplications, Topics Appl. Phys.  103 , 351–366 (2006) © Springer-Verlag Berlin Heidelberg 2006  352 Vladimir P. Drachev and Vladimir M. Shalaev Fig. 1.  Typical protein spot on ASF substrate before ( a ) and after ( b ) washingwith a Tris-buffered saline solution (the 2 µ l aliquot of 0 . 5 µ M bacterial alkalinephosphatase/C-terminal FLAG-peptide fusion (fBAP) was deposited manually),spot size is about 2mm; ( c ) – antibody array deposited with a quill-type spotter,typical size of about 4 × 4mm, individual spot size is about 100 µ m portunity is enabled with SERS detection and a pseudotyping procedure forEbola virus, a potential biowarfare agent. Antibody–antigen binding resultsin distinct spectral changes in SERS spectra of the first layer protein, eitherantibody or antigen, depending on the chosen binding scheme. While thisChapter addresses SERS applications, we should mention the excellent per-formance of adaptive surfaces as a solid support for antibody–antigen bindingreactions tested with a microarray protocol and fluorescence detection andcompared with commercial substrates [19,21]. 2 Adaptive Plasmonic Nanostructures Both SERS enhancement mechanisms – electromagnetic and molecular (chem-ical) – work effectively for molecules in immediate proximity to a rough metalsurface. Hence molecule adsorption is an important component of SERS andis always accompanied by surface-chemistry processes. One of the key ideasbehind adaptive plasmonic nanostructures is to provide the needed flexibilityunder protein deposition for surface-chemistry to form particle aggregatesnaturally covered and stabilized with the proteins of interest.Figure 1a shows a typical protein spot after manual deposition of proteinsin a Tris-buffered saline (TBS) solution on an ASF with subsequent drying.In the example in the figure, the concentration of the deposited antibody was1 µ M and the volume was about 2 µ l. After washing with TBS/Tween-20 for15min to 30min, the nonadherent metal is removed from the substrate exceptin the areas where protein (antibody or antigen) has been deposited (Fig. 1b).An example of an array deposition with a quill-type spotter is shown inFig. 1c. The capture proteins (a set contains antihuman interleukin 6 mono-clonal antibody and other) were dissolved in phosphate-buffered saline (PBS)(300 µ g / ml) for spotting on the ASF substrate. The approximate spotting vol-ume was 0 . 7nl, yielding spots of about 100 µ m in diameter. The substrateswere custom printed by Tele-Chem International.The nanostructures of the ASF before and after protein deposition andwashing are shown in Fig. 2a and b. The initial film was fabricated with an e-beam evaporator at high vacuum (10 − 7 Torr); clean glass slides were covered  Biomolecule Sensing with Adaptive Plasmonic Nanostructures 353 Fig. 2.  FE SEM images of ASF substrates: ( a ) initial structure of 11nm silverfilm ( b ) same substrate as in ( a ) but inside antibody spot after deposition of 2 µ l,0 . 5 µ M in TBS solution and washing; ( c ) substrate after deposition of TBS withoutproteins; ( d ) absorbance inside protein spot (antihuman interleukin 10) at 568nmversus protein concentration. ( a ) and ( b ) are adapted with permission from [19] © 2005 American Chemical Society first by a sublayer of 10nm of SiO 2  followed by an 8nm to 11nm Ag layerdeposited at a rate of 0 . 05nm / s. As illustrated by the field emission scan-ning electron microscope (FE SEM) images, protein-mediated restructuringresults in the formation of aggregates of silver particles covered with proteins(Fig. 2b), as opposed to the relatively disintegrated but closely spaced parti-cles of the initial film before protein deposition (Fig. 2a). Depending on themass thickness of the initial film, small or large fractal-like aggregates can beformed during the nanoscale restructuring process.A lower concentration of protein results in lower metal coverage (the ra-tio of white area to total area in the FE SEM images) and lower extinctionat a particular wavelength. A decrease of metal coverage correlates with de-creasing optical absorption. The absorbance of the metal film inside a proteinspot increases linearly with protein concentration and then saturates above acertain concentration, which can be considered to be optimal (Fig. 2d). Theconcentration dependence shows almost no change after 30min of washing inTBS/Tween 20 solution, which confirms the stabilization of the film by theproteins.The transparent areas outside of a typical protein spot certainly containno silver particles, as indicated by absorption measurements in those areas.The metal-particle coverage inside the protein spot is also reduced relativeto the initial film. To determine the chemical form of the silver remaining  354 Vladimir P. Drachev and Vladimir M. Shalaev in the transparent areas, TBS (Tris: 0 . 05M, NaCl: 0 . 138M, KCl: 0 . 0027M)was deposited on the ASF and dried. Then the deposition region was studiedwith FE SEM and X-ray diffraction analysis. The transparent area contains40  ×  40 µ m particles as seen from FE SEM images (Fig. 2c). X-ray diffrac-tion results indicate a reduced Ag 101 peak and two peaks from NaCl andAgCl. This implies that silver particles are transformed to silver salt in thetransparent area.An estimate of the thermodynamics of the (redox) reaction of Ag withoxygen shows that Ag oxidation is a downhill reaction (with negative freeenergy) under the experimental conditions. When the silver film is exposedto a TBS buffer (pH 7.4), metal silver tends to be oxidized by oxygen andform AgCl due to the low solubility of AgCl in water ( K  sp  = 1 . 8 × 10 − 10 ) [22].The total reaction and its standard potential under standard conditions [22]will be:4Ag+4Cl − +O 2 +4H + = 4AgCl+2H 2 O , E  ◦ =  E  ox ◦ − E  red ◦ = 1 . 007V . For calculating the actual reaction potential under the experimental conditionat 298K, we use the Nernst equation: E   =  E  ◦ −  (0 . 059 / 4)log { 1 / ([Cl − ] 4 [H + ] 4 P O 2 } , where [Cl − ] is about 0 . 14M, [H + ] is about 10 − 7 . 4 M and the partial pressureof oxygen in air is 0 . 21 atm. So the experimental value of   E   is estimatedto be 0 . 51V, which means that the reaction is thermodynamically favorable(downhill in free energy). The positive potential is the driving force for theoxidation of Ag to Ag + .Thus a deposition of proteins in a buffer solution results in the competitionof two processes: etching through the oxidation of silver surface in buffer, andstabilization with protein interaction. Etching of the particle surface makesparticles movable and leads to the protein-mediated rearrangement of theinitial particle nanostructure. 3 SERS Features of Conformational States: Insulin Insulin represents an interesting example for Raman spectroscopy. It consistsof 51 aminoacids distributed in two chains (the A and B chains), which arelinked by two disulfide bonds. Insulin typically exists in the hexameric form,although its monomeric form is the active form of the hormone. The size of aninsulin hexamer is about 3nm to 5nm. The problem of insulin oligomeriza-tion has stimulated development of a number of recombinant insulin analogs.The first of these molecules, insulin  lispro,  is engineered as a rapidly act-ing, blood-glucose-lowering agent. ASFs were used to examine the differencesin Raman spectra of two insulin isomers: human insulin and insulin lispro.Both of these molecules have the same set of amino acid side chains, and  Biomolecule Sensing with Adaptive Plasmonic Nanostructures 355 differ only in the two amino acid residue locations, causing a slight changein the molecules’ conformational states. Specifically, the lysine and prolineresidues on the C-terminal of the B chain are interchanged in their positionsin lispro molecules as compared to human insulin. This small difference, how-ever, causes an important clinical effect for diabetes treatment, making insulinlispro a fast-acting agent in the bloodstream while human insulin is slower toact. The differences in SERS spectra between these two insulin isomers canbe detected with ASFs and assigned to the  α -helix Raman markers and Phering-breathing mode.The Raman system used in this study comprises an Ar/Kr ion laser(Melles Griot), a laser bandpath holographic filter, two Super-Notch Plusfilters (Kaiser Optical Systems), focusing and collection lenses, an Acton Re-search 300i monochromator with a grating of 1200 grooves per millimeter,and a liquid-nitrogen-cooled CCD system (1340 × 400 pixels, Roper Scien-tific). SERS spectra were typically collected using an excitation laser wave-length of 568 . 2nm with normal incidence and 45 ◦ scattering. An objectivelens (f/1.6) provided a collection area of about 180 µ m 2 . The spectral resolu-tion was about 3cm − 1 . Normal Raman spectra were collected in a backscat-tering geometry using a micro-Raman system, which consists of a He–Ne laser(632 . 8nm) with 12mW power focused to a spot of about 2 µ m diameter onthe sample using a 80 ×  microscope objective. 3.1 SERS Versus Normal Raman A SERS spectrum collected from the central part of an insulin spot is shownin Fig. 3a after linear polynomial background subtraction and normalization.The spot was deposited from a 3 µ l drop of 1 µ M insulin in a 0 . 1mM HClsolution. The extinction coefficient  ε 280  of 5 . 7mM − 1 · cm − 1 [23] and Ramanband assignments are known from the published literature [24,25].A comparison of the insulin SERS spectra with our normal Raman spec-tra of insulin on quartz (Fig. 3b) and with insulin in solution [24] suggeststhat all the Raman fingerprints of insulin are enhanced by approximately thesame factor in our study. Phenylalanine (Phe) and tyrosine (Tyr) often con-tribute to the protein Raman spectra along with the amide I and amide IIIbands of the peptide backbone vibrations. Insulin contains phenylalanine lo-cated at the B1, B24, B25 residues of the B chain and tyrosine at A14, A19,B16, and B26 [25]. The spectra contain “indicators” of the phenylalanine-to-tyrosine ratio: two peaks at 624 (Phe) and 643 (Tyr) cm − 1 ; the pair of tyrosine Raman makers at 832 and 850cm − 1 , the backbone-related C α –Cstretching mode at 945cm − 1 ; and phenylalanine ring modes at 1003cm − 1 and 1030cm − 1 . In addition there are a group of peaks between 1150cm − 1 and 1500cm − 1 that can be constituted from Tyr and Phe peaks at 1176cm − 1 and 1206cm − 1 , amide III bands at 1242cm − 1 and 1267cm − 1 (they givethe peak at 1248cm − 1 , Fig. 3b), –CH deformation modes at 1308cm − 1 and1342cm − 1 (peak at 1323cm − 1 in our case), a –COO − symmetrical stretching
Search
Related Search
We Need Your Support
Thank you for visiting our website and your interest in our free products and services. We are nonprofit website to share and download documents. To the running of this website, we need your help to support us.

Thanks to everyone for your continued support.

No, Thanks
SAVE OUR EARTH

We need your sign to support Project to invent "SMART AND CONTROLLABLE REFLECTIVE BALLOONS" to cover the Sun and Save Our Earth.

More details...

Sign Now!

We are very appreciated for your Prompt Action!

x